Pulmonary endothelial cells are a rich source of nitric oxide (NO) which[unreadable] is generated from L-arginine by the endothelial isoform of NO synthase[unreadable] (eNOS). NO production by endothelial cells can be increased by[unreadable] extracellular L-arginine despite a saturating a intracellular arginine[unreadable] concentration for eNOS. This observation has been termed the "arginine[unreadable] paradox" and can not be explained based on available data. We hypothesize[unreadable] that a caveolar complex between eNOS and the cationic amino acid[unreadable] transporter (CAT-1) responsible for arginine transport exists in lung[unreadable] endothelial cells. Such a complex provides an efficient mechanism for the[unreadable] directed delivery of substrate to eNOS and would account for the "arginine[unreadable] paradox". Since caveolae interact with the cytoskeleton through actin-[unreadable] associated proteins and microtubules, we also hypothesize that L-arginine[unreadable] transport is regulated by membrane-cytoskeleton interactions. Finally,[unreadable] based on work from the PI's laboratory, we hypothesize that hypoxia[unreadable] inhibits L-arginine transport by disrupting membrane-cytoskeleton[unreadable] interactions through a calpain-mediated mechanism. To verify that the CAT-[unreadable] 1 transporter is localized to caveolae in lung endothelial cells, we will[unreadable] use immunohistochemistry and deconvolution fluorescence microscopy to[unreadable] localize CAT-1, and we will isolate caveolae and identify the presence of[unreadable] the CAT-1 transporter by immunoblot analysis and transport assays. We will[unreadable] also use immunohistochemistry and deconvolution microscopy to determine[unreadable] whether CAT-1 and eNOS co-localize caveolae. We will confirm the existence[unreadable] of a caveolar complex by immunodepletion assays and fractionation of CAT-[unreadable] 1-eNOS protein complexes on sucrose gradients. To define membrane[unreadable] cytoskeletal interactions that affect CAT-1-mediated L-arginine transport,[unreadable] we will selectively disrupt the actin-microfilament and microtubule[unreadable] systems in lung endothelial cells and then measure CAT-1 mediated[unreadable] transport. We will identify qualitative and quantitative changes in[unreadable] cytoskeletal elements responsible for alterations in arginine transport.[unreadable] Finally, we will use immunohistochemistry, deconvolution fluorescence[unreadable] microscopy, and immunoblot analyses to assess the effect of hypoxia on the[unreadable] cytoskeleton and arginine transport with special emphasis on the role of[unreadable] calpain in mediating the hypoxic effects. These studies will advance our[unreadable] understanding of the mechanisms that regulate arginine transport and NO[unreadable] production in lung endothelial cells and will ultimately lead to new and[unreadable] improve ways to attenuate or prevent pulmonary vascular dysfunction.[unreadable]